Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Drs. Harvey A. Goldberg

2nd Supervisor

Frank Beier

Joint Supervisor

Abstract

The mechanism of biomineralization during endochondral ossification is an incompletely characterized process. The small integrin-binding ligand N-linked glycoprotein (SIBLING) group of proteins are anionic phosphoproteins found in the extracellular matrix of mineralized tissues and have been postulated to modulate mineral growth. Bone sialoprotein (BSP) has been shown to be a promoter of both osteoblastic differentiation and mineralization. In contrast, osteopontin (OPN) has been shown to be an inhibitor of mineralization. This thesis examined the phenotype of mineralization upon loss of either BSP or OPN. When the Bsp gene was ablated in mice (Bsp-/-) histological analyses revealed tibiae had delayed endochondral ossification, decreased mineralization, and shortened length. Additionally, the mice had altered growth plates with decreased chondrocyte proliferation and apoptosis, possibly contributing to the shortened bones. Studies on Bsp-/- osteogenic cultures agreed with the in vivo findings, demonstrating delayed osteogenic differentiation and mineralization. Adenoviral-mediated overexpression of BSP in Bsp-/- osteogenic cultures increased osteogenic gene expression and mineralization. Specific mutations of BSP that truncated the N-terminus, mutated poly-E to poly-A, and RGD integrin-binding to KAE, all resulted in increased mineralization and osteogenic gene expression. Loss of any one functional motif did not result in loss of functionality of BSP. Deletion of Opn (Opn-/-) in murine-derived osteogenic cultures resulted in increased mineralization with no physiological change in osteoblast gene expression. Supplementation with milk OPN and OPN-derived peptides reduced the enhanced mineralization of the Opn-/- osteoblasts without altering their iv terminal differentiation characteristics. These studies demonstrate that the SIBLING proteins are potent mediators of mineralization.

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